Little is known about how and why hypoxia arises in tumors, ie. whether hypoxia is a chronic process resulting from diffusion limitations, or occurs more acutely due to transient changes in blood perfusion. A new technique for isolating viable tumor cells as a function of their oxygenation status, recently reported by us, shows a clear role of acute hypoxia in at least one experimental tumor system. We now propose to systematically study several additional transplantable murine tumors using this technique. Intravenous administration of the flurochrome Hoechst 33342 into tumor bearing mice results in a heterogeneous staining pattern within the tumors determined by stain delivery (blood flow), and cell location (cells nearer functional vessels are more intensely stained). These differences in staining persist after tumor disaggregation, thus permitting isolation of differentially stained cell populations by fluorescence activated cell sorting. Cells sorted using these techniques will be analyzed for viability using a soft agar clonogenic assay. By analyzing the radiation response of cell populations exposed to Hoechst 33342 either during or at various times before irradiation, we expect to identify acutely and chronically hypoxic cells. The effects of tumor type, size and site of implantation on the existence and amount of acute hypoxia will be investigated. Independent histological procedures will be used to determine the opening and closing rates of individual blood vessels. Reoxygenation and repair of radiation damage in acutely or chronically hypoxic tumor cells will also be evaluated, as will the effectiveness of treatment modalities such as perfluorocarbons, carbogen breathing, and hyperbaric oxygen designed to elminate hypoxia. These studies will lead to a better understanding of tumor hypoxia and its influence on radiation response, a question of particular importance since treatment modalities designed specifically to overcome chronic hypoxia may not be as effective against acutely hypoxic cells.